Production of lipase

ABSTRACT

A lipase enzyme which hydrolyzes glycerides of long-chain carboxylic acids in preference to those of short-chain carboxylic acids can be produced by growing under aerobic conditions a culture of a strain of Absidia in a medium containing appropriate nutrients and then recovering the enzyme therefrom. An especially useful strain is Absidia butleri.

United States Patent [72] inventors Nicholas Melachouris;

Robert Lemoyne Charles, both of Elkhart, Ind.

[21] Appl. No. 856,149

[22] Filed Sept. 8, 1969 [45] Patented Jan. 11, 1972 [73] Assignee MilesLaboratories, Inc.

Elkhart, Ind.

[54] PRODUCTION OF LIPASE [50] Field of Search 195/66, 62

[56] References Cited UNITED STATES PATENTS 3,256,157 6/1966 Truant eta1. i. 195/62 3,431,175 3/1969 Arima et a1. 195/62 PrimaryExaminer-Lionel M. Shapiro Att0rneys-Joseph C. Schwalbach, Michael A.Kondzella,

Louis E. Davidson and Harry T. Stephenson ABSTRACT: A lipase enzymewhich hydrolyzes glycerides of long-chain carboxylic acids in preferenceto those of shortchain carboxylic acids can be produced by growing underaerobic conditions a culture ofa strain of Absidia in a mediumcontaining appropriate nutrients and then recovering the enzymetherefrom. An especially useful strain is Absidia butleri.

PRODUCTION OF LIPASE BACKGROUND OF THE INVENTION Lipases are knownenzymes having the capability of catalyzing the hydrolysis of fats, suchas glycerides of fatty acids. Generally, these lipases are extractedfrom various plant sources, such as from castor beans, and from animalsources, such as from the pancreas. 'These prior art lipases generallyhave the property of preferentially hydrolyzing glycerides ofshort-chain carboxylic acids having about 4 to carbon atoms in the acidchain as contrasted to hydrolysis of glycerides of long-chain carboxylicacids having about 14 to carbon atoms in the acid chain.

SUMMARY OF THE INVENTION In accordance with the present invention, aprocess is provided for producing a lipase comprising growing underaerobic conditions a culture of a strain of Absidia in a mediumcontaining appropriate nutrients and then recovering the enzymetherefrom. A novel lipase is thus produced which hydrolyzes glyceridesof long-chain carboxylic acids in preference to hydrolyzing glyceridesof short-chain carboxylic acids.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In carrying out the process ofthe present invention a strain of Absidia is grown in a suitable medium.Exemplary useful strains are Absidia .butleri, Absidia cylindrosporavar. rhizomorpha, and Absidia blakesleeana. The preferred stain isAbsidia butleri. An especially useful strain of Absidia butleri isolatedfrom soil has been deposited with the Northern Utilization ResearchBranch, Agricultural Research Service of the United States Department ofAgriculture, Peoria, Illinois and has been given the identificationnumber NRRL 3521. This culture is available to the public withoutrestriction. While Absidia butleri NRRL 3521 is the preferred strain, itis understood that natural and artificial mutants of Absidia butleriNRRL 352i can also be employed. Such mutants can be obtained by wellknown techniques such as X-ray and ultraviolet irradiation.

The strains useful in the present invention are maintained on agarslants in a well-known manner and can be grown in a medium containingsources of carbohydrates and nitrogen. Preferably the medium alsocontains inorganic salts. Illustrative carbohydrates are corn starch,corn dextrin, dextrose, lactose, milk solids and the like, Illustrativenitrogen compounds are soy meal, protein containing materials, aminoacids, yeast extract, cottonseed flour, meat meal and the like.Illustrative inorganic salts are calcium carbonate, zinc sulfate,monosodium phosphate, disodium phosphate and the like. Thesecarbohydrates, nitrogen sources and inorganic salts are well known inthe art. It is preferable that the medium contain ground corn as asource of some of the carbohydrates, nitrogen and inorganic salts. It isalso preferable that the medium contain bacterial amylase to aid inliquefaction of the carbohydrates.

The Absidia strain is preferably grown under submerged conditions forabout 3 to about 7 days at a temperature of from about to about 40 C. Attemperatures below about 25 C. the yield of desired enzyme is too low tobe economically practical, while at temperatures above about 40 C. thedesired enzyme becomes inactivated. The preferred growth temperature isfrom about 32+ to about 37 C. The growth medium should also preferablyhave a pH of from about 4.8 to about 8.2.

The desired lipase enzyme of the present invention is present in thebeer outside the mycelium and can be recovered in the liquid form bysimply filtering ofi the myceli um from the fennentation beer andretaining the filtrate. The enzyme can be recovered in a powdered form,if desired, by well-known enzyme recovery techniques. The filtrate canbe frozen and dried from the frozen state using known freeze-dryingtechniques. The fermentation beer filtrate could alternatively betreated with ethanol to precipitate the enzyme. The precipitated enzymeis then washed with ethanol and vacuum dried at room temperature.Acetone or other organic solvents, as well as ammonium sulfate, can alsobe used to precipitate the enzyme from an aqueous solution.

The lipase enzyme produced by the present process is assayed forlipolytic activity by the following method.

LIPASE ACTIVITY A reaction mixture is prepared by blending 5 ml. of a 10weight percent emulsion of a carboxylic acid glyceride in 10 weightpercent aqueous solution of gum arabic, 2 ml. of a 0.001 M aqueoussolution of tris(hydroxymethyl)aminomethane-HC1 buffer at pH 8.0, 2 ml.of 3.0 M aqueous sodium chloride solution, 1 ml. of 0.03 M aqueouscalcium chloride solution and 5 ml. distilled water. The above totalreaction mixture is adjusted to pH 8.0 and placed in the reaction wellof an instrument which is capable of adding suffcient quantities of 0.02N aqueous sodium hydroxide to maintain the pH of the reaction mixture at8.0. The enzyme to be assayed, if a powder, is dissolved in 0.001 Maqueous solution of tris-(hydroxymethyl)aminomethane-HC I buffer at pH8.0. If a liquid enzyme is to be assayed, it is adjusted to pH 8.0. A 2ml. portion of the liquid enzyme sample is added to the above reactionmixture and the reaction mixture-enzyme blend is maintained at 35 C. for34 minutes. During this time the pH of the total blend is maintained at8.0. The total elapsed time in seconds and the total amount of 0.02 Naqueous sodium hydroxide added to maintain pH 8.0 are measured. Thelipase activity is then calculated as follows:

Lipase Units V l ,200/T wherein:

V=Volume of sodium hydroxide (ml.)

T=Time (sec) The enzyme activity is then expressed in Lipase Units (LU)per ml. of liquid sample or per mg. of dried sample. A Lipase Unit isdefined as the amount of enzyme required to produce one micromole ofcarboxylic acid per min. under the conditions specified. The sodiumhydroxide added to maintain pH neutralizes the carboxylic acid producedand is a measure of such carboxylic acid production. Lipase activitytoward shortchain carboxylic acid glycerides is measured usingtributyrin as the substrate. It contains four carbon atoms in itscarboxylic acid chain. Lipase activity toward long chain carboxylic acidglycerides is measured using butteroil (about l4 to 20 carbon atoms inits major carboxylic acid chain constituents) and olive oil (about 18carbon atoms in its major carboxylic acid chain constituent).

The present invention will be further described in the followingillustrative examples.

EXAMPLE 1 A culture of Absidia butleri NRRL 3521 was transferred toseveral l-liter Erlenmeyer flasks each containing a sterilized mediumconsisting of 3.2 g. dextrose monohydrate, 5.0 g. ground corn, 2.9 g. ofan amino acid mixture marketed under the trade name Peptone TW, 502 mg.monosodium phosphate, 824 mg. disodium phosphate, 40 mg. bacterialamylase and ml. water. The pH of this medium was 6.4. The inoculatedflask contents were then shaken on a reciprocal motion shaker with a2-inch stroke at 228 strokes per min. for 6 days while maintained at atemperature of 33 C. The resulting fermentation beer was then filteredfrom the mycelium, the combined filtrate from all flasks was freezedried and assayed for lipase activity using several substrates. Thelipase activity is shown in table 1 below. The lipase activity of thenovel enzyme of the present invention is also compared to the activityof a prior art lipase, steapsin.

TABLE 1 Lipase Activity (LU/mg.)

Substrate Enzyme Tributyrin Butteroil Olive Oil A. butleri Steapsin Itcan be seen from the above table that the lipase activity of the lipaseproduced in accordance with the present invention toward glycerides oflong-chain carboxylic acids is about four times that of its activitytoward glycerides of short-chain carboxylic acids. In contrast to thisthe prior art lipase has an acl0 tivity toward glycerides of short-chaincarboxylic acids which is about two to four times that of its activitytoward glycerides of long-chain carboxylic acids. This preferentialactivity of the lipase of the present invention provides improved lipaseutility when included in detergent compositions and in medicated creams,for example. cl EXAMPLE 2 A culture of Absidia butleri NRRL 3521 wastransferred to an aerated fermentor containing a sterilized mediumconsisting of 100 pounds corn dextrin, 50 pounds soy meal, 25 poundscalcium carbonate, 570 g. dextrose monohydrate, 31.16 g. zinc sulfatemonohydrate and 300 gal. water. The pH of this medium was 8.1. Theculture was grown at 32 C. for 6 days. The resulting fermentation beerwas then filtered from the mycelium and an aliquot portion of thefiltrate was freezedried and assayed for lipase activity using severalsubstrates. The lipase activity is shown in table 2 below.

TABLE 2 Lipase Activity (LU/mg.)

Substrate Buttcroil Enzyme Tributyrin Olive Oil A. butlcri 0.073 0.3570.323

EXAMPLE 3 um phosphate, 412 mg. disodium phosphate, 40 mg. bacterialamylase and ml. water. The pH of this medium was 6.45. The inoculatedflask contents were then shaken on a reciprocal motion shaker with a2-inch stroke at 228 strokes per minute for 6 days at a temperature of33 C. The resulting fermentation beer was then filtered from themycelium. The lipase activity of the enzymes produced from each culturestrain was then assayed using several substrates. The results are shownin table 3 below.

TABLE 3 Lipase Activity (LU/ml.)

Substrate Enzyme Tributyrin Bntteroil Olive Oil A. cylindrospora var.rhizomorpha NRRL 2815 0.086 0.505 0.]57 A. blakcslccana NRRL I305 0.3780.437 0.516

sidia is Absidia butleri NRRL 3521 or mutants thereof.

4. A process according to claim 1 wherern the straln of Absidia isAbsidia cylindrospora var. rhizomorpha.

5. A process according to claim 1 wherein the strain of Absidia isAbsidia blakesleeana.

6. A lipase enzyme produced by the process of claim 1 which hydrolyzesglycerides of long chain carboxylic acids in preference to glycerides ofshort chain carboxylic acids.

2. A process according to claim 1 wherein the strain of Absidia isAbsidia butleri.
 3. A process according to claim 1 wherein the strain ofAbsidia is Absidia butleri NRRL 3521 or mutants thereof.
 4. A processaccording to claim 1 wherein the strain of Absidia is Absidiacylindrospora var. rhizomorpha.
 5. A process according to claim 1wherein the strain of Absidia is Absidia blakesleeana.
 6. A lipaseenzyme produced by the process of claim 1 which hydrolyzes glycerides oflong chain carboxylic acids in preference to glycerides of short chaincarboxylic acids.